Methods of treating glucose metabolism disorders

ABSTRACT

Methods of treating individuals with a glucose metabolism disorder, and compositions thereof, are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority benefit of U.S. provisional applicationSer. No. 61/304,264, filed Feb. 12, 2010, which application isincorporated herein by reference in its entirety.

INTRODUCTION

High blood glucose levels stimulate the secretion of insulin bypancreatic beta-cells. Insulin in turn stimulates the entry of glucoseinto muscles and adipose cells, leading to the storage of glycogen andtriglycerides and to the synthesis of proteins. Activation of insulinreceptors on various cell types diminishes circulating glucose levels byincreasing glucose uptake and utilization, and by reducing hepaticglucose output. Disruptions within this regulatory network can result indiabetes and associated pathologic syndromes that affect a large andgrowing percentage of the human population.

Patients who have a glucose metabolism disorder can suffer fromhyperglycemia, hyperinsulinemia, and/or glucose intolerance. An exampleof a disorder that is often associated with the aberrant levels ofglucose and/or insulin is insulin resistance, in which fliver, fat, andmuscle cells lose their ability to respond to normal blood insulinlevels.

Therapy that can modulate glucose and/or insulin levels in a patient andto enhance the biological response to fluctuating glucose levels remainsof interest.

SUMMARY OF THE INVENTION

The present disclosure provides compositions that find use in modulatingglucose and/or insulin levels in glucose metabolism disorders. Thepresent methods involve using an isolated protein FAM3C (Family withsequence similarity 3, member C) for modulating glucose metabolism. Theprotein may be used as therapy to treat various glucose metabolismdisorders, such as diabetes mellitus, and/or obesity. The subjectproteins encompass those expressed by FAM3C genes, and homologuesthereof, and are useful for but not limited to treating one or more ofthe following conditions: diabetes mellitus (e.fg. diabetes type I,diabetes type II and gestational diabetes), insulin resistance,hyperinsulinemia, glucose intolerance, hyperglycemia or metabolicsyndrome.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows body weight of mice on a high fat diet that were injectedwith an adeno-associated virus (AAV) expressing a protein of the presentdisclosure (mouse ortholog) compared to those of mice injected with acontrol virus and those on a lean diet (n=5 mice per group).

FIG. 2 shows blood glucose of mice on high fat diet that were injectedwith AAV expressing a protein of the present disclosure (mouse ortholog)compared to those of mice injected with a control virus and those on alean diet (n=5 mice per group).

FIG. 3 shows insulin levels of mice on high fat diet that were injectedwith AAV expressing a protein of the present disclosure (mouse ortholog)compared to those of mice injected with a control virus and those on alean diet (n=5 mice per group).

FIG. 4 shows the level of glucose in mice over a 60 minute period postinjection of 1 g/kg of glucose. Glucose tolerance was monitored in miceon a high fat diet that have been injected with AVV expressing a proteinprovided by the present disclosure (mouse ortholog) or the control andin mice that were on a lean diet (n=5 mice per group).

FIG. 5 shows the result of an insulin tolerance test. Glucose levelswere monitored after an intraparitoneal injection of insulin (0.75units/kg). Response to insulin was compared among DIO mice injected withAAV expressing a protein of the present disclosure (mouse ortholog) andthose injected with AAV expressing the control, as well as lean mice(n=5 mice per group)

FIG. 6 shows body weight of mice on a high fat diet that were injectedwith AAV expressing a protein of the present disclosure (human ortholog)compared to those of mice injected with a control virus and those on alean diet (n=5 mice per group).

FIG. 7 shows blood glucose of mice on high fat diet that were injectedwith AAV expressing a protein of the present disclosure (human ortholog)compared to those of mice injected with a control virus and those on alean diet (n=5 mice per group).

FIG. 8 shows insulin levels of mice on high fat diet that were injectedwith AAV expressing a protein of the present disclosure (human ortholog)compared to those of mice injected with a control virus and those on alean diet (n=5 mice per group).

FIG. 9 shows the level of glucose in mice over a 60 minute period postinjection of 1 g/kg of glucose. Glucose tolerance was monitored in miceon a high fat diet that have been injected with AVV expressing a proteinprovided by the present disclosure (human ortholog) compared to those ofmice injected with a control virus, as well as lean mice (n=5 mice pergroup).

FIG. 10 shows the result of an insulin tolerance test. Glucose levelswere monitored after an intraperitoneal injection of insulin (0.75units/kg). Response to insulin was compared among diet-induced obesity(DIO) mice injected with AAV expressing a protein of the presentdisclosure (human ortholog) and those injected with AAV expressing thecontrol (n=5 mice per group).

FIG. 11 shows an alignment of various amino acid sequences of FAM3C.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “theprotein” includes reference to one or more proteins, and so forth. It isfurther noted that the claims may be drafted to exclude any optionalelement. As such, this statement is intended to serve as antecedentbasis for use of such exclusive terminology as “solely,” “only” and thelike in connection with the recitation of claim elements, or use of a“negative” limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

Overview

The present disclosure provides compositions that find use in modulatingglucose and/or insulin levels in glucose metabolism disorders. Thecompositions encompass FAM3C (Family with sequence similarity 3, memberC), genes and/or proteins encoded thereby, and are useful for but notlimited to treating diabetes mellitus (e.g. diabetes type I, diabetestype II, and gestational diabetes). In a diet-induced obesity model(mice on a high fat diet), the glucose and insulin levels are higherthan those in a subject on a regular lean diet. However, when treatedwith AAV expressing the proteins of the present disclosure, the subjecton the high fat diet regains the ability to regulate glucose levels, toan extent seen in subjects on a regular lean diet. Accordingly, theproteins of the present disclosure may be used in restoring glucosehomeostasis in subjects with a dysfunctional glucose metabolism,including subjects who may be overweight, obese, and/or on a high fatdiet.

Definitions

The terms “patient” or “subject” as used interchangeably herein in thecontext of therapy, refer to a human and non-human animal, as therecipient of a therapy or preventive care.

The phrase “in a sufficient amount to effect a change in” means thatthere is a detectable difference between a level of an indicatormeasured before and after administration of a particular therapy.Indicators include but are not limited to glucose and insulin.

The phrase “glucose tolerance”, as used herein, refers to the ability ofa subject to control the level of plasma glucose and/or plasma insulinwhen glucose intake fluctuates. For example, glucose toleranceencompasses the ability to reduce the level of plasma glucose back to alevel before the intake of glucose within about 120 minutes or so.

The phrase “pre-diabetes”, as used herein, refers a condition that maybe determined using either the fasting plasma glucose test (FPG) or theoral glucose tolerance test (OGTT). Both require a person to fastovernight. In the FPG test, a person's blood glucose is measured firstthing in the morning before eating. In the OGTT, a person's bloodglucose is checked after fasting and again 2 hours after drinking aglucose-rich drink. In a healthy individual, a normal test result of FPGwould indicate a glucose level of below about 100 mg/dl. A subject withpre-diabetes would have a FPG level between about 100 and about 125mg/dl. If the blood glucose level rises to about 126 mg/dl or above, thesubject is determined to have “diabetes”. In the OGTT, the subject'sblood glucose is measured after a fast and 2 hours after drinking aglucose-rich beverage. Normal blood glucose in a healthy individual isbelow about 140 mg/dl 2 hours after the drink. In a pre-diabeticsubject, the 2-hour blood glucose is about 140 to about 199 mg/dl. Ifthe 2-hour blood glucose rises to 200 mg/dl or above, the subject isdetermined to have “diabetes”.

“FAM3C” (Family with sequence similarity 3, member C), also known as“GS3786” or “ILEI” (“interleukin-related protein interleukin-like EMTinducer”), encompasses murine and human proteins that are encoded bygene FAM3C or a gene homologue of FAM3C. FAM3C is found in many mammals(e.g. human, non-human primates, canines, and mouse). See FIG. 11 foralignments of various amino acid sequences of FAM3C.

As used herein, “homologues” or “variants” refers to protein or DNAsequences that are similar based on their amino acid or nucleic acidsequences, respectively. Homologues or variants encompass naturallyoccurring DNA sequences and proteins encoded thereby and their isoforms.The homologues also include known allelic or splice variants of aprotein/gene. Homologues and variants also encompass nucleic acidsequences that vary in one or more bases from a naturally-occurring DNAsequence but still translate into an amino acid sequence that correspondto the naturally-occurring protein due to degeneracy of the geneticcode. Homologues and variants may also refer to those that differ fromthe naturally-occurring sequences by one or more conservativesubstitutions and/or tags and/or conjugates.

The terms “polypeptide,” “peptide,” and “protein”, used interchangeablyherein, refer to a polymeric form of amino acids of any length, whichcan include genetically coded and non-genetically coded amino acids,chemically or biochemically modified or derivatized amino acids, andpolypeptides having modified peptide backbones. The term includes fusionproteins, including, but not limited to, fusion proteins with aheterologous amino acid sequence, fusions with heterologous andhomologous leader sequences, with or without N-terminal methionineresidues; immunologically tagged proteins; and the like.

It will be appreciated that throughout this present disclosure referenceis made to amino acids according to the single letter or three lettercodes. For the reader's convenience, the single and three letter aminoacid codes are provided below:

G Glycine Gly A Alanine Ala L Leucine Leu M Methionine Met FPhenylalanine Phe W Tryptophan Trp K Lysine Lys Q Glutamine Gln EGlutamic Acid Glu S Serine Ser P Proline Pro V Valine Val I IsoleucineIle C Cysteine Cys Y Tyrosine Tyr H Histidine His R Arginine Arg NAsparagine Asn D Aspartic Acid Asp T Threonine Thr

The terms “nucleic acid molecule” and “polynucleotide” are usedinterchangeably and refer to a polymeric form of nucleotides of anylength, either deoxyribonucleotides or ribonucleotides, or analogsthereof. Non-limiting examples of polynucleotides include linear andcircular nucleic acids, messenger RNA (mRNA), cDNA, recombinantpolynucleotides, vectors, probes, and primers.

The term “heterologous” refers to two components that are defined bystructures derived from different sources. For example, where“heterologous” is used in the context of a polypeptide, where thepolypeptide includes operably linked amino acid sequences that can bederived from different polypeptides (e.g., a first component consistingof a recombinant peptide and a second component derived from a nativeFAM3C polypeptide). Similarly, “heterologous” in the context of apolynucleotide encoding a chimeric polypeptide includes operably linkednucleic acid sequence that can be derived from different genes (e.g., afirst component from a nucleic acid encoding a peptide according to anembodiment disclosed herein and a second component from a nucleic acidencoding a carrier polypeptide). Other exemplary “heterologous” nucleicacids include expression constructs in which a nucleic acid comprising acoding sequence is operably linked to a regulatory element (e.g., apromoter) that is from a genetic origin different from that of thecoding sequence (e.g., to provide for expression in a host cell ofinterest, which may be of different genetic origin relative to thepromoter, the coding sequence or both). For example, a T7 promoteroperably linked to a polynucleotide encoding a FAM3C polypeptide ordomain thereof is said to be a heterologous nucleic acid. “Heterologous”in the context of recombinant cells can refer to the presence of anucleic acid (or gene product, such as a polypeptide) that is of adifferent genetic origin than the host cell in which it is present.

The term “operably linked” refers to functional linkage betweenmolecules to provide a desired function. For example, “operably linked”in the context of nucleic acids refers to a functional linkage betweennucleic acids to provide a desired function such as transcription,translation, and the like, e.g., a functional linkage between a nucleicacid expression control sequence (such as a promoter, signal sequence,or array of transcription factor binding sites) and a secondpolynucleotide, wherein the expression control sequence affectstranscription and/or translation of the second polynucleotide. “Operablylinked” in the context of a polypeptide refers to a functional linkagebetween amino acid sequences (e.g., of different domains) to provide fora described activity of the polypeptide.

As used herein in the context of the structure of a polypeptide,“N-terminus” and “C-terminus” refer to the extreme amino and carboxylends of the polypeptide, respectively, while “N-terminal” and“C-terminal” refer to relative positions in the amino acid sequence ofthe polypeptide toward the N-terminus and the C-terminus, respectively,and can include the residues at the N-terminus and C-terminus,respectively. “Immediately N-terminal” or “immediately C-terminal”refers to a position of a first amino acid residue relative to a secondamino acid residue where the first and second amino acid residues arecovalently bound to provide a contiguous amino acid sequence.

“Derived from” in the context of an amino acid sequence orpolynucleotide sequence (e.g., an amino acid sequence “derived from” aFAM3C polypeptide) is meant to indicate that the polypeptide or nucleicacid has a sequence that is based on that of a reference polypeptide ornucleic acid (e.g., a naturally occurring FAM3C polypeptide orFAM3C-encoding nucleic acid), and is not meant to be limiting as to thesource or method in which the protein or nucleic acid is made.

“Isolated” refers to a protein of interest that, if naturally occurring,is in an environment different from that in which it may naturallyoccur. “Isolated” is meant to include proteins that are within samplesthat are substantially enriched for the protein of interest and/or inwhich the protein of interest is partially or substantially purified.Where the protein is not naturally occurring, “isolated” indicates theprotein has been separated from an environment in which it was made byeither synthetic or recombinant means.

“Enriched” means that a sample is non-naturally manipulated (e.g., by anexperimentalist or a clinician) so that a protein of interest is presentin a greater concentration (e.g., at least a three-fold greater, atleast 4-fold greater, at least 8-fold greater, at least 64-fold greater,or more) than the concentration of the protein in the starting sample,such as a biological sample (e.g., a sample in which the proteinnaturally occurs or in which it is present after administration), or inwhich the protein was made (e.g., as in a bacterial protein and thelike).

“Substantially pure” indicates that an entity (e.g., polypeptide) makesup greater than about 50% of the total content of the composition (e.g.,total protein of the composition) and typically, greater than about 60%of the total protein content. More typically, a “substantially pure”refers to compositions in which at least 75%, at least 85%, at least 90%or more of the total composition is the entity of interest (e.g. 95%, ofthe total protein. Preferably, the protein will make up greater thanabout 90%, and more preferably, greater than about 95% of the totalprotein in the composition.

FAM3C

The subject proteins find use in regulating levels of glucose andinsulin in a subject. Such proteins find use in treating and/orpreventing aberrant levels of glucose and insulin, even if the subjecthas or has been on a high-fat diet.

The present disclosure provides the use of proteins encompassingnaturally-occurring full-length and/or fragments of an amino acidsequence of a FAM3C polypeptide and homologues from different species,and use of such proteins in preparation of formulation for therapy andin methods of treating glucose imbalance in a patient. Exemplaryembodiments of such are described below.

“FAM3C”, as used in the method of the present disclosure is also knownas “family with sequence similarity 3, member C”. FAM3C encompassesmurine and human variants that are encoded by the FAM3C gene or a genehomologous to FAM3C.

FAM3C refers to FAM3C proteins or FAM3C DNA sequences, which encompasstheir naturally occurring isoforms and/or allelic/splice variants. AFAM3C protein also refers to proteins that have one or more alterationin the amino acid residues (e.g. at locations that are not conservedacross variants and/or species) while retaining the conserved domainsand having the same biological activity as the naturally-occurringFAM3C. FAM3C also encompasses nucleic acid sequences that vary in one ormore bases from a naturally-occurring DNA sequence but still translateinto an amino acid sequence that correspond to the a naturally-occurringprotein due to degeneracy of the genetic code. For example, FAM3C mayalso refer to those that differ from the naturally-occurring sequencesof FAM3C by one or more conservative substitutions and/or tags and/orconjugates.

Proteins used in the method of the present disclosure contain contiguousamino acid residues of a length derived from FAM3C. A sufficient lengthof contiguous amino acid residues may vary depending on the specificnaturally-occurring amino acid sequence from which the protein isderived. For example, the protein may be at least 100 amino acids to 150amino acid residues in length, at least 150 amino acids to 200 aminoacid residues in length, or at least 220 amino acids up to thefull-length protein (e.g., 223 amino acids, 224 amino acids, 227 aminoacids). For example, the protein may be of about 224 amino acid residuesin length when derived from a human FAM3C protein, or of about 223 aminoacid residues in length when derived from a mouse FAM3C protein.

A protein containing an amino acid sequence that is substantiallysimilar to the amino acid sequence of a FAM3C polypeptide includes apolypeptide comprising an amino acid sequence having at least about 71%,at least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 95%, at least about 98%, or at least about99%, amino acid sequence identity to a contiguous stretch of from about100 amino acids (aa) to about 150 aa, from about 150 aa to about 200 aa,from about 200 aa to about 220 aa, or from about 220 aa up to the fulllength of a naturally occurring FAM3C polypeptide. For example, a FAM3Cpolypeptide suitable for use in a subject method can comprise an aminoacid sequence having at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 94%, at least about 95%,at least about 98%, or at least about 99%, amino acid sequence identityto a contiguous stretch of from about 100 amino acids (aa) to about 150aa, from about 150 aa to about 200 aa, from about 200 aa to about 220aa, or from about 220 aa up to the full length, of the human FAM3Cpolypeptide amino acid sequence depicted in FIG. 11.

The protein may lack at least 5, at least 10, up to at least 50 or moreaa relative to a naturally-occurring full-length FAM3C polypeptide. Forexample, the protein may not contain the signal sequence of based on theamino acid sequence of a naturally-occurring FAM3C polypeptide. Theprotein may also contain the same or similar glycosylation pattern asthose of a naturally-occurring FAM3C polypeptide, may contain noglycosylation, or the glycosylation pattern of host cells used toproduce the protein.

Many DNA and protein sequences of FAM3C are known in the art and certainsequences are discussed later below.

The proteins used in the method of the present disclosure include thosecontaining contiguous amino acid sequences of any naturally-occurringFAM3C, as well as those having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 usuallyno more than 20, 10, or 5 amino acid substitutions, where thesubstitution is usually a conservative amino acid substitution. By“conservative amino acid substitution” generally refers to substitutionof amino acid residues within the following groups:

1) L, I, M, V, F;

2) R, K;

3) F, Y, H, W, R;

4) G, A, T, S;

5) Q, N; and

6) D, E.

Conservative amino acid substitutions in the context of a peptide or aprotein disclosed herein are selected so as to preserve putativeactivity of the protein. Such presentation may be preserved bysubstituting with an amino acid with a side chain of similar acidity,basicity, charge, polarity, or size to the side chain of the amino acidbeing replaced. Guidance for substitutions, insertion, or deletion maybe based on alignments of amino acid sequences of different variantproteins or proteins from different species. For example, according tothe alignment shown in FIG. 11, at certain residue positions that arefully conserved (*), substitution, deletion or insertion may not beallowed while at other positions where one or more residues are notconserved, an amino acid change can be tolerated. Residues that aresemi-conserved (. or :) may tolerate changes that preserve charge,polarity, and/or size.

The present disclosure provides any of the FAM3C polypeptides describedabove. The protein may be isolated from a natural source, e.g., is in anenvironment other than its naturally-occurring environment. The subjectprotein may also be recombinantly made, e.g., in a genetically modifiedhost cell (e.g., bacteria; yeast; Pichia; insect; mammalian cells; andthe like), where the genetically modified host cell is geneticallymodified with a nucleic acid comprising a nucleotide sequence encodingthe subject protein. The subject protein encompasses syntheticpolypeptides, e.g., a subject synthetic polypeptide is synthesizedchemically in a laboratory (e.g., by cell-free chemical synthesis).Methods of productions are described in more detail below.

Nucleic Acid and Protein Sequences

The subject polypeptide may be generated using recombinant techniques tomanipulate nucleic acids of different FAM3C known in the art to provideconstructs encoding a protein of interest. It will be appreciated that,provided an amino acid sequence, the ordinarily skilled artisan willimmediately recognize a variety of different nucleic acids encoding suchamino acid sequence in view of the knowledge of the genetic code.

For production of subject protein derived from naturally-occurringpolypeptides, it is noted that nucleic acids encoding a variety ofdifferent FAM3C polypeptides are known and available in the art. Nucleicacid (and amino acid sequences) for various FAM3C are also provided inGenBank as accession nos.: 1) Homo sapiens: amino acid sequenceNP_(—)055703.1; nucleotide sequence: NM_(—)014888.2; 2) Mus musculus:amino acid sequence NP_(—)613053.3; nucleotide sequence NM_(—)138587.4;3) Rattus norvegicus: amino acid sequence NP_(—)942066.1; nucleotidesequence NM_(—)198771.1; 4) Bos taurus: amino acid sequenceNP_(—)001092617; nucleotide sequence NM_(—)001099147. Exemplary aminoacid sequences are depicted in FIG. 11. Several sequences and furtherinformation on the nucleic acid and protein sequences can also be foundin the Example section below.

It will be appreciated that the nucleotide sequences encoding theprotein may be modified so as to optimize the codon usage to facilitateexpression in a host cell of interest (e.g., Escherichia. coli, and thelike). Methods for production of codon optimized sequences are known inthe art.

Protein Modifications

The proteins used in the present disclosure can be provided as proteinsthat are modified relative to the naturally-occurring protein. Purposesof the modifications may be to increase a property desirable in aprotein formulated for therapy (e.g. serum half-life), to raise antibodyfor use in detection assays, and/or for protein purification, and thelike.

One way to modify a subject protein is to conjugate (e.g. link) one ormore additional elements at the N- and/or C-terminus of the protein,such as another protein (e.g. having an amino acid sequence heterologousto the subject protein) and/or a carrier molecule. Thus, an exemplaryprotein can be provided as fusion proteins with a polypeptide(s) derivedfrom a FAM3C polypeptide.

Conjugate modifications to proteins may result in a protein that retainsthe desired activity, while exploiting properties of the second moleculeof the conjugate to impart and/or enhances certain properties (e.g.desirable for therapeutic uses). For example, the polypeptide may beconjugated to a molecule, e.g., to facilitate solubility, storage,half-life, reduction in immunogenicity, controlled release in tissue orother bodily location (e.g., blood or other particular organs, etc.).

Other features of a conjugated protein may include one where theconjugate reduces toxicity relative to unconjugated protein. Anotherfeature is that the conjugate may target a type of cell or organ moreefficiently than an unconjugated material. The protein can optionallyhave attached a drug to further counter the causes or effects associatedwith disorders of glucose metabolism (e.g., drug for high cholesterol),and/or can optionally be modified to provide for improvedpharmacokinetic profile (e.g., by PEGylation, hyperglycosylation, andthe like).

Modifications that can enhance serum half-life of the subject proteinsare of interest. A subject protein may be “PEGylated”, as containing oneor more poly(ethylene glycol) (PEG) moieties. Methods and reagentssuitable for PEGylation of a protein are well known in the art and maybe found in U.S. Pat. No. 5,849,860, disclosure of which is incorporatedherein by reference. PEG suitable for conjugation to a protein isgenerally soluble in water at room temperature, and has the generalformula R(O—CH₂—CH₂)_(n)O—R, where R is hydrogen or a protective groupsuch as an alkyl or an alkanol group, and where n is an integer from 1to 1000. Where R is a protective group, it generally has from 1 to 8carbons.

The PEG conjugated to the subject protein can be linear. The PEGconjugated to the subject protein may also be branched. Branched PEGderivatives such as those described in U.S. Pat. No. 5,643,575,“star-PEG's” and multi-armed PEG's such as those described in ShearwaterPolymers, Inc. catalog “Polyethylene Glycol Derivatives 1997-1998.” StarPEGs are described in the art including, e.g., in U.S. Pat. No.6,046,305.

Where the proteins are to be incorporated into a liposome, carbohydrate,lipid moiety, including N-fatty acyl groups such as N-lauroyl, N-oleoyl,fatty amines such as dodecyl amine, oleoyl amine, and the like (e.g.,see U.S. Pat. No. 6,638,513) may also be used to modify the subjectproteins.

Where the subject proteins are used to raise antibodies specific for thesubject protein, elements that may be conjugated include large, slowlymetabolized macromolecules such as: proteins; polysaccharides, such assepharose, agarose, cellulose, cellulose beads and the like; polymericamino acids such as polyglutamic acid, polylysine, and the like; aminoacid copolymers; inactivated virus particles; inactivated bacterialtoxins such as toxoid from diphtheria, tetanus, cholera, leukotoxinmolecules; liposomes; inactivated bacteria; dendritic cells; and thelike.

Additional suitable carriers used in eliciting antibodies are well knownin the art, and include, e.g., thyroglobulin, albumins such as humanserum albumin, tetanus toxoid; Diphtheria toxoid; polyamino acids suchas poly(D-lysine:D-glutamic acid); VP6 polypeptides of rotaviruses;influenza virus hemagglutinin, influenza virus nucleoprotein; hepatitisB virus core protein, hepatitis B virus surface antigen; purifiedprotein derivative (PPD) of tuberculin from Mycobacterium tuberculosis;inactivated Pseudomonas aeruginosa exotoxin A (toxin A); Keyhole LimpetHemocyanin (KLH); filamentous hemagglutinin (FHA) of Bordetellapertussis; T helper cell (Th) epitopes of tetanus toxoid (TT) andBacillus Calmette-Guerin (BCG) cell wall; recombinant 10 kDa, 19 kDa and30-32 kDa proteins from M. leprae or from M. tuberculosis, or anycombination of these proteins; and the like. See, e.g., U.S. Pat. No.6,447,778 for a discussion of carriers, and for methods of conjugatingpeptides to carriers.

Where the subject protein is to be isolated from a source, the subjectprotein can be conjugated to moieties the facilitate purification, suchas members of specific binding pairs, e.g., biotin (member ofbiotin-avidin specific binding pair), an antibody, a lectin, and thelike. A subject protein can also be bound to (e.g., immobilized onto) asolid support, including, but not limited to, polystyrene plates orbeads, magnetic beads, test strips, membranes, and the like.

Where the proteins are to be detected in an assay, the subject proteinsmay also contain a detectable label, e.g., a radioisotope (e.g., ¹²⁵I;³⁵S, and the like), an enzyme which generates a detectable product(e.g., luciferase, β-galactosidase, horse radish peroxidase, alkalinephosphatase, and the like), a fluorescent protein, a chromogenicprotein, dye (e.g., fluorescein isothiocyanate, rhodamine,phycoerythrin, and the like); fluorescence emitting metals, e.g., ¹⁵²Eu,or others of the lanthanide series, attached to the protein throughmetal chelating groups such as EDTA; chemiluminescent compounds, e.g.,luminol, isoluminol, acridinium salts, and the like; bioluminescentcompounds, e.g., luciferin; fluorescent proteins; and the like. Indirectlabels include antibodies specific for a subject protein, wherein theantibody may be detected via a secondary antibody; and members ofspecific binding pairs, e.g., biotin-avidin, and the like.

Any of the above elements that are used to modify the subject proteinsmay be linked to the polypeptide via a linker, e.g. a flexible linker.Where a subject protein is a fusion protein comprising a FAM3Cpolypeptide and a heterologous fusion partner polypeptide, a subjectfusion protein can have a total length that is equal to the sum of theFAM3C polypeptide and the heterologous fusion partner polypeptide.

Linkers suitable for use in modifying the proteins of the presentdisclosure include “flexible linkers”. If present, the linker moleculesare generally of sufficient length to permit the protein and a linkedcarrier to allow some flexible movement between the protein and thecarrier. The linker molecules are generally about 6-50 atoms long. Thelinker molecules may also be, for example, aryl acetylene, ethyleneglycol oligomers containing 2-10 monomer units, diamines, diacids, aminoacids, or combinations thereof. Other linker molecules which can bind topolypeptides may be used in light of this disclosure.

Suitable linkers can be readily selected and can be of any of a suitableof different lengths, such as from 1 amino acid (e.g., Gly) to 20 aminoacids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.

Exemplary flexible linkers include glycine polymers (G)_(n),glycine-serine polymers (including, for example, (GS)_(n), GSGGS_(n)(SEQ ID NO: 1) and GGGS_(n) (SEQ ID NO: 2), where n is an integer of atleast one), glycine-alanine polymers, alanine-serine polymers, and otherflexible linkers known in the art. Glycine and glycine-serine polymersare of interest since both of these amino acids are relativelyunstructured, and therefore may serve as a neutral tether betweencomponents. Glycine polymers are of particular interest since glycineaccesses significantly more phi-psi space than even alanine, and is muchless restricted than residues with longer side chains (see Scheraga,Rev. Computational Chem. 11173-142 (1992)). Exemplary flexible linkersinclude, but are not limited GGSG (SEQ ID NO:3), GGSGG (SEQ ID NO:4),GSGSG (SEQ ID NO: 5), GSGGG (SEQ ID NO: 6), GGGSG (SEQ ID NO: 7), GSSSG(SEQ ID NO: 8), and the like. The ordinarily skilled artisan willrecognize that design of a peptide conjugated to any elements describedabove can include linkers that are all or partially flexible, such thatthe linker can include a flexible linker as well as one or more portionsthat confer less flexible structure.

Methods of Production

The proteins of the present disclosure can be produced by any suitablemethod, including recombinant and non-recombinant methods (e.g.,chemical synthesis). Where a polypeptide is chemically synthesized, thesynthesis may proceed via liquid-phase or solid-phase. Solid-phasesynthesis (SPPS) allows the incorporation of unnatural amino acidsand/or peptide/protein backbone modification. Various forms of SPPS,such as Fmoc and Boc, are available for synthesizing peptides of thepresent invention. Details of the chemical synthesis are known in theart (e.g. Ganesan A. 2006 Mini Rev. Med Chem. 6:3-10 and Camarero J A etal. 2005 Protein Pept Lett. 12:723-8). Briefly, small insoluble, porousbeads are treated with functional units on which peptide chains arebuilt. After repeated cycling of coupling/deprotection, the freeN-terminal amine of a solid-phase attached is coupled to a singleN-protected amino acid unit. This unit is then deprotected, revealing anew N-terminal amine to which a further amino acid may be attached. Thepeptide remains immobilized on the solid-phase and undergoes afiltration process before being cleaved off.

Where the protein is produced using recombinant techniques, the proteinsmay be produced as an intracellular protein or as a secreted protein,using any suitable construct and any suitable host cell, which can be aprokaryotic or eukaryotic cell, such as a bacterial (e.g. E. coli) or ayeast host cell, respectively.

Other examples of eukaryotic cells that may be used as host cellsinclude insect cells, mammalian cells, and/or plant cells. Wheremammalian host cells are used, the cells may include one or more of thefollowing: human cells (e.g. HeLa, 293, H9 and Jurkat cells); mousecells (e.g., NIH3T3, L cells, and C127 cells); primate cells (e.g. Cos1, Cos 7 and CV1) and hamster cells (e.g., Chinese hamster ovary (CHO)cells).

A wide range of host-vector systems suitable for the expression of thesubject protein may be employed according standard procedures known inthe art. See for example, Sambrook et al. 1989 Current Protocols inMolecular Biology Cold Spring Harbor Press, New York and Ausubel et al.1995 Current Protocols in Molecular Biology, Eds. Wiley and Sons.

Methods for introduction of genetic material into host cells include,for example, transformation, electroporation, conjugation, calciumphosphate methods and the like. The method for transfer can be selectedso as to provide for stable expression of the introduced FAM3C-encodingnucleic acid. The polypeptide-encoding nucleic acid can be provided asan inheritable episomal element (e.g., plasmid) or can be genomicallyintegrated. A variety of appropriate vectors for use in production of apolypeptide of interest are available commercially.

Vectors can provide for extrachromosomal maintenance in a host cell orcan provide for integration into the host cell genome. The expressionvector provides transcriptional and translational regulatory sequences,and may provide for inducible or constitutive expression, where thecoding region is operably linked under the transcriptional control ofthe transcriptional initiation region, and a transcriptional andtranslational termination region. In general, the transcriptional andtranslational regulatory sequences may include, but are not limited to,promoter sequences, ribosomal binding sites, transcriptional start andstop sequences, translational start and stop sequences, and enhancer oractivator sequences. Promoters can be either constitutive or inducible,and can be a strong constitutive promoter (e.g., T7, and the like).

Expression constructs generally have convenient restriction siteslocated near the promoter sequence to provide for the insertion ofnucleic acid sequences encoding proteins of interest. A selectablemarker operative in the expression host may be present to facilitateselection of cells containing the vector. In addition, the expressionconstruct may include additional elements. For example, the expressionvector may have one or two replication systems, thus allowing it to bemaintained in organisms, for example in mammalian or insect cells forexpression and in a prokaryotic host for cloning and amplification. Inaddition the expression construct may contain a selectable marker geneto allow the selection of transformed host cells. Selectable genes arewell known in the art and will vary with the host cell used.

Isolation and purification of a protein can be accomplished according tomethods known in the art. For example, a protein can be isolated from alysate of cells genetically modified to express the proteinconstitutively and/or upon induction, or from a synthetic reactionmixture, by immunoaffinity purification, which generally involvescontacting the sample with an anti-protein antibody, washing to removenon-specifically bound material, and eluting the specifically boundprotein. The isolated protein can be further purified by dialysis andother methods normally employed in protein purification methods. In oneembodiment, the protein may be isolated using metal chelatechromatography methods. Protein of the present disclosure may containmodifications to facilitate isolation, as discussed above.

The subject proteins may be prepared in substantially pure or isolatedform (e.g., free from other polypeptides). The protein can present in acomposition that is enriched for the polypeptide relative to othercomponents that may be present (e.g., other polypeptides or other hostcell components). Purified protein may be provided such that the proteinis present in a composition that is substantially free of otherexpressed proteins, e.g., less than 90%, usually less than 60% and moreusually less than 50% of the composition is made up of other expressedproteins.

Compositions

The present disclosure provides compositions comprising a subjectprotein, which may be administered to a subject in need of restoringglucose homeostasis.

A subject protein composition can comprise, in addition to a subjectprotein, one or more of: a salt, e.g., NaCl, MgCl, KCl, MgSO₄, etc.; abuffering agent, e.g., a Tris buffer,N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES),2-(N-Morpholino)ethanesulfonic acid (MES),2-(N-Morpholino)ethanesulfonic acid sodium salt (MES),3-(N-Morpholino)propanesulfonic acid (MOPS),N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; asolubilizing agent; a detergent, e.g., a non-ionic detergent such asTween-20, etc.; a protease inhibitor; glycerol; and the like.

Compositions comprising a subject protein may include a buffer, which isselected according to the desired use of the protein, and may alsoinclude other substances appropriate to the intended use. Those skilledin the art can readily select an appropriate buffer, a wide variety ofwhich are known in the art, suitable for an intended use.

The composition may comprise a pharmaceutically acceptable excipient, avariety of which are known in the art and need not be discussed indetail herein. Pharmaceutically acceptable excipients have been amplydescribed in a variety of publications, including, for example,“Remington: The Science and Practice of Pharmacy”, 19^(th) Ed. (1995),or latest edition, Mack Publishing Co; A. Gennaro (2000) “Remington: TheScience and Practice of Pharmacy”, 20th edition, Lippincott, Williams, &Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C. Ansel et al., eds 7^(th) ed., Lippincott, Williams, & Wilkins; andHandbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al., eds.,3^(rd) ed. Amer. Pharmaceutical Assoc.

A subject pharmaceutical composition can comprise a FAM3C polypeptide(e.g., a purified FAM3C polypeptide), and a pharmaceutically acceptableexcipient. In some cases, a subject pharmaceutical composition will besuitable for injection into a subject, e.g., will be sterile. Forexample, in some embodiments, a subject pharmaceutical composition willbe suitable for injection into a human subject, e.g., where thecomposition is sterile and is free of detectable pyrogens and/or othertoxins.

The protein compositions may comprise other components, such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium,carbonate, and the like. The compositions may contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions such as pH adjusting and buffering agents, toxicity adjustingagents and the like, for example, sodium acetate, sodium chloride,potassium chloride, calcium chloride, sodium lactate, hydrochloride,sulfate salts, solvates (e.g., mixed ionic salts, water, organics),hydrates (e.g., water), and the like.

For example, compositions may include aqueous solution, powder form,granules, tablets, pills, suppositories, capsules, suspensions, sprays,and the like. The composition may be formulated according to thedifferent routes of administration described later below.

Where the protein is administered as an injectable (e.g. subcutaneously,intraperitoneally, and/or intravenous) directly into a tissue, aformulation can be provided as a ready-to-use dosage form, or asnon-aqueous form (e.g. a reconstitutable storage-stable powder) oraqueous form, such as liquid composed of pharmaceutically acceptablecarriers and excipients. The protein-containing formulations may also beprovided so as to enhance serum half-life of the subject proteinfollowing administration. For example, the protein may be provided in aliposome formulation, prepared as a colloid, or other conventionaltechniques for extending serum half-life. A variety of methods areavailable for preparing liposomes, as described in, e.g., Szoka et al.1980 Ann. Rev. Biophys. Bioeng. 9:467, U.S. Pat. Nos. 4,235,871,4,501,728 and 4,837,028. The preparations may also be provided incontrolled release or slow-release forms.

Other examples of formulations suitable for parenteral administrationinclude isotonic sterile injection solutions, anti-oxidants,bacteriostats, and solutes that render the formulation isotonic with theblood of the intended recipient, suspending agents, solubilizers,thickening agents, stabilizers, and preservatives. For example, asubject pharmaceutical composition can be present in a container, e.g.,a sterile container, such as a syringe. The formulations can bepresented in unit-dose or multi-dose sealed containers, such as ampulesand vials, and can be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid excipient, forexample, water, for injections, immediately prior to use. Extemporaneousinjection solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described.

The concentration of the subject proteins in a formulation can varywidely (e.g., from less than about 0.1%, usually at or at least about 2%to as much as 20% to 50% or more by weight) and will usually be selectedprimarily based on fluid volumes, viscosities, and patient-based factorsin accordance with the particular mode of administration selected andthe patient's needs.

Patient Populations

The present disclosure provides a method to treat a patient sufferingfrom hyperglycemia, hyperinsulinemia, and/or glucose intolerance. Suchconditions are also commonly associated with many other glucosemetabolism disorders. As such, patients of glucose metabolism disorderscan be candidates for therapy according to the subject methods.

The phrase “glucose metabolism disorder” encompasses any disordercharacterized by a clinical symptom or a combination of clinicalsymptoms that are associated with an elevated level of glucose and/or anelevated level of insulin in a subject relative to a healthy individual.Elevated levels of glucose and/or insulin may be manifested in thefollowing disorders and/or conditions: type II diabetes (e.g.insulin-resistance diabetes), gestational diabetes, insulin resistance,impaired glucose tolerance, hyperinsulinemia, impaired glucosemetabolism, pre-diabetes, metabolic disorders (such as metabolicsyndrome which is also referred to as syndrome X), obesity,obesity-related disorder.

An example of a suitable patient may be one who is hyperglycemic and/orhyperinsulinemic and who is also diagnosed with diabetes mellitus (e.g.Type II diabetes). “Diabetes” refers to a progressive disease ofcarbohydrate metabolism involving inadequate production or utilizationof insulin and is characterized by hyperglycemia and glycosuria.

“Hyperglycemia”, as used herein, is a condition in which an elevatedamount of glucose circulates in the blood plasma relative to a healthyindividual and can be diagnosed using methods known in the art. Forexample, hyperglycemia can be diagnosed as having a fasting bloodglucose level between 5.6 to 7 mM (pre-diabetes), or greater than 7 mM(diabetes).

“Hyperinsulinemia”, as used herein, is a condition in which there areelevated levels of circulating insulin while blood glucose levels mayeither be elevated or remain normal. Hyperinsulininemia can be caused byinsulin resistance which is associated with dyslipidemia such as hightrglycerides, high cholesterol, high LDL and low HDL, high uric acids,polycystic ovary syndrome, type II diabetes and obesity.Hyperinsulinemia can be diagnosed as having a plasma insulin levelhigher than about 2 μU/mL.

A patient having any of the above disorders may be a suitable candidatein need of a therapy in accordance with the present method so as toreceive treatment for hyperglycemia, hyperinsulinemia, and/or glucoseintolerance. Administering the subject protein in such an individual canrestore glucose homeostasis and may also decrease one or more ofsymptoms associated with the disorder.

Candidates for treatment using the subject method may be determinedusing diagnostic methods known in the art, e.g. by assaying plasmaglucose and/or insulin levels. Candidates for treatment include thosewho have exhibited or are exhibiting higher than normal levels of plasmaglucose/insulin. Such patients include patients who have a fasting bloodglucose concentration (where the test is done after 8 to 10 hour fast)of higher than about 100 mg/dL, e.g., higher than about 110 mg/dL,higher than about 120 mg/dL, about 150 mg/dL up to about 200 mg/dL ormore. Individuals suitable to be treated also include those who have a 2hour postprandial blood glucose concentration or a concentration after aglucose tolerance test (e.g. 2 hours after ingestion of a glucose-richdrink), in which the concentration is higher than about 140 mg/dL, e.g.,higher than about 150 mg/dL up to 200 mg/dL or more. Glucoseconcentration may also be presented in the units of mmol/L, which can beacquired by dividing mg/dL by a factor of 18.

Methods

The subject method involves administering the subject proteins in asubject who has hyperglycemia, hyperinsulinemia, and/or glucoseintolerance. The methods of the present disclosure include administeringFAM3C (polypeptide or nucleic acid) in the context of a variety ofconditions including glucose metabolism disorders, including theexamples above (in both prevention and post-diagnosis therapy).

Subjects having, suspected of having, or at risk of developing a glucosemetabolism disorder are contemplated for therapy and diagnosis describedherein.

By “treatment” it is meant that at least an amelioration of the symptomsassociated with the condition afflicting the host is achieved, whereamelioration refers to at least a reduction in the magnitude of aparameter, e.g. symptom, associated with the condition being treated. Assuch, treatment includes situations where the condition, or at leastsymptoms associated therewith, are reduced or avoided. Thus treatmentincludes: (i) prevention, that is, reducing the risk of development ofclinical symptoms, including causing the clinical symptoms not todevelop, e.g., preventing disease progression to a harmful or otherwiseundesired state; (ii) inhibition, that is, arresting the development orfurther development of clinical symptoms, e.g., mitigating or completelyinhibiting an active disease (e.g., so as to decrease level of insulinand/or glucose in the bloodstream, to increase glucose tolerance so asto minimize fluctuation of glucose levels, and/or so as to protectagainst diseases caused by disruption of glucose homeostasis).

In the methods of the present disclosure, protein compositions describedherein can be administered to a subject (e.g. a human patient) to, forexample, achieve and/or maintain glucose homeostasis, e.g., to reduceglucose level in the bloodstream and/or to reduce insulin level to arange found in a healthy individual. Subjects for treatment includethose having a glucose metabolism disorder as described herein. Forexample, protein composition finds use in facilitating glucosehomeostasis in subjects with a glucose metabolism disorder resultingfrom obesity.

The methods relating to disorders of the glucose metabolism contemplatedherein include, for example, use of protein described above for therapyalone or in combination with other types of therapy. The method involvesadministering to a subject the subject protein (e.g. subcutaneously orintravenously). As noted above, the methods are useful in the context oftreating or preventing a wide variety of disorders related to glucosemetabolism.

Routes of Administration

In practicing the methods, routes of administration (path by which asubject protein is brought into a subject) may vary. A subject proteinabove can be delivered by a route that provides for delivery of theprotein to the bloodstream (e.g., by parenteral administration, such asintravenous administration, intramuscular administration, and/orsubcutaneous administration). Injection can be used to accomplishparenteral administration.

Combination Therapy

Any of a wide variety of therapies directed to regulating glucosemetabolism, and any glucose metabolism disorders, and/or obesity, forexample, can be combined in a composition or therapeutic method with thesubject proteins. The subject proteins can also be administered incombination with a modified diet and/or exercise regiment to promoteweight loss.

“Combination” as used herein is meant to include therapies that can beadministered separately, e.g. formulated separately for separateadministration (e.g., as may be provided in a kit), or undertaken as aseparate regime (as in exercise and diet modifications), as well as foradministration in a single formulation (i.e., “co-formulated”). Examplesof agents that may be provided in a combination therapy include thosethat are normally administered to subjects suffering from symptoms ofhyperglycemia, hyperinsulinemia, glucose intolerance, and disordersassociated those conditions. Examples of agents that may be provided ina combination therapy include those that promote weight loss.

Where the subject protein is administered in combination with one ormore other therapies, the combination can be administered anywhere fromsimultaneously to up to 5 hours or more, e.g., 10 hours, 15 hours, 20hours or more, prior to or after administration of a subject protein. Incertain embodiments, a subject protein and other therapeuticintervention are administered or applied sequentially, e.g., where asubject protein is administered before or after another therapeutictreatment. In yet other embodiments, a subject protein and other therapyare administered simultaneously, e.g., where a subject protein and asecond therapy are administered at the same time, e.g., when the secondtherapy is a drug it can be administered along with a subject protein astwo separate formulations or combined into a single composition that isadministered to the subject. Regardless of whether administeredsequentially or simultaneously, as illustrated above, the treatments areconsidered to be administered together or in combination for purposes ofthe present disclosure.

Additional standard therapeutics for glucose metabolism disorders thatmay or may not be administered in conjunction with a subject protein,include but not limited to any of the combination therapies describedabove, hormonal therapy, immunotherapy, chemotherapeutic agents andsurgery.

Dosages

In the methods, a therapeutically effective amount of a subject proteinis administered to a subject in need thereof. For example, a subjectprotein causes the level of plasma glucose and/or insulin to return to anormal level relative to a healthy individual when the subject proteinis delivered to the bloodstream in an effective amount to a patient whopreviously did not have a normal level of glucose/insulin relative to ahealthy individual prior to being treated. The amount administeredvaries depending upon the goal of the administration, the health andphysical condition of the individual to be treated, age, the degree ofresolution desired, the formulation of a subject protein, the activityof the subject proteins employed, the treating clinician's assessment ofthe medical situation, the condition of the subject, and the body weightof the subject, as well as the severity of the dysregulation ofglucose/insulin and the stage of the disease, and other relevantfactors. The size of the dose will also be determined by the existence,nature, and extent of any adverse side-effects that might accompany theadministration of a particular protein.

It is expected that the amount will fall in a relatively broad rangethat can be determined through routine trials. For example, the amountof subject protein employed to restore glucose homeostasis is not morethan about the amount that could otherwise be irreversibly toxic to thesubject (i.e., maximum tolerated dose). In other cases, the amount isaround or even well below the toxic threshold, but still in an effectiveconcentration range, or even as low as threshold dose.

Also, suitable doses and dosage regimens can be determined bycomparisons to indicators of glucose metabolism. Such dosages includedosages which result in the stabilized levels of glucose and insulin,for example, comparable to a healthy individual, without significantside effects. Dosage treatment may be a single dose schedule or amultiple dose schedule (e.g., including ramp and maintenance doses). Asindicated below, a subject composition may be administered inconjunction with other agents, and thus doses and regiments can vary inthis context as well to suit the needs of the subject.

Individual doses are typically not less than an amount required toproduce a measurable effect on the subject, and may be determined basedon the pharmacokinetics and pharmacology for absorption, distribution,metabolism, and excretion (“ADME”) of the subject protein or itsby-products, and thus based on the disposition of the composition withinthe subject. This includes consideration of the route of administrationas well as dosage amount, which can be adjusted for enteral (applied viadigestive tract for systemic or local effects when retained in part ofthe digestive tract) or parenteral (applied by routes other than thedigestive tract for systemic or local effects) applications. Forinstance, administration of a subject protein is typically via injectionand often intravenous, intramuscular, or a combination thereof.

By “therapeutically effective amount” is meant that the administrationof that amount to an individual, either in a single dose, as part of aseries of the same or different protein compositions, is effective tohelp restore homeostasis of glucose metabolism as assessed by glucoseand/or insulin levels in a subject. As noted above, the therapeuticallyeffective amount can be adjusted in connection with dosing regimen anddiagnostic analysis of the subject's condition (e.g., monitoring for thelevels of glucose and/or insulin in the plasma) and the like.

As an example, the effective amount of a dose or dosing regimen can begauged from the ED₅₀ of a protein for inducing an action that leads toclearing glucose from the bloodstream or lowering of insulin levels. By“ED₅₀” (effective dosage) is the intended dosage which induces aresponse halfway between the baseline and maximum after some specifiedexposure time. The ED₅₀ of a graded dose response curve thereforerepresents the concentration of a subject protein where 50% of itsmaximal effect is observed. ED₅₀ may be determined by in vivo studies(e.g. animal models) using methods known in the art.

An effective amount may not be more than 100× the calculated ED₅₀. Forinstance, the amount of protein that is administered is less than about100×, less than about 50×, less than about 40×, 35×, 30×, or 25× andmany embodiments less than about 20×, less than about 15× and even lessthan about 10×, 9×, 8×, 7×, 6×, 5×, 4×, 3×, 2× or 1× than the calculatedED₅₀. In one embodiment, the effective amount is about 1× to 30× of thecalculated ED₅₀, and sometimes about 1× to 20×, or about 1× to 10× ofthe calculated ED₅₀. In other embodiments, the effective amount is thesame as the calculated ED₅₀, and in certain embodiments the effectiveamount is an amount that is more than the calculated ED₅₀.

An effective amount of a protein may also an amount that is effective,when administered in one or more doses, to reduce in an individual alevel of plasma glucose and/or plasma insulin that is elevated relativeto that of a healthy individual by at least about 10%, at least about20%, at least about 25%, at least about 30%, at least about 40%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, or more than 80%, compared to an elevated level of plasmaglucose/insulin in the individual not treated with the protein.

Further examples of dose per administration may be at less than 10 μg,less than 2 μg, or less than 1 μg. Dose per administration may also bemore than 50 μg, more 100 μg, more than 300 μg to 600 μg or more. Anexample of a range of dosage per weight is about 0.1 μg/kg to about 1μg/kg, up to about 1 mg/kg or more. Effective amounts and dosage regimencan readily be determined empirically from assays, from safety andescalation and dose range trials, individual clinician-patientrelationships, as well as in vitro and in vivo assays known in the art.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of proteins ofthe present disclosure calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms depend on the particular protein employed and the effect tobe achieved, and the pharmacodynamics associated with each protein inthe host.

Kits

Also provided by the present disclosure are kits for using thecompositions disclosed herein and for practicing the methods, asdescribed above. The kits may be provided for administration of thesubject protein in a subject in need of restoring glucose homeostasis.The kit can include one or more of the proteins disclosed herein, whichmay be provided in a sterile container, and can be provided informulation with a suitable a pharmaceutically acceptable excipient foradministration to a subject. The proteins can be provided with aformulation that is ready to be used as it is or can be reconstituted tohave the desired concentrations. Where the proteins are provided to bereconstituted by a user, the kit may also provide buffers,pharmaceutically acceptable excipient, and the like, packaged separatelyfrom the subject protein. The proteins of the present kit may beformulated separately or in combination with other drugs. Kits caninclude, for example: 1) a first container (e.g., a sterile container)comprising a subject pharmaceutical composition; and 2) a secondcontainer (e.g., a sterile container) comprising a second agent (e.g., asecond agent that can lower blood glucose levels).

In addition to above-mentioned components, the kits can further includeinstructions for using the components of the kit to practice the subjectmethods. The instructions for practicing the subject methods aregenerally recorded on a suitable recording medium. For example, theinstructions may be printed on a substrate, such as paper or plastic,etc. As such, the instructions may be present in the kits as a packageinsert, in the labeling of the container of the kit or componentsthereof (i.e., associated with the packaging or subpackaging) etc. Inother embodiments, the instructions are present as an electronic storagedata file present on a suitable computer readable storage medium, e.g.CD-ROM, diskette, etc. In yet other embodiments, the actual instructionsare not present in the kit, but means for obtaining the instructionsfrom a remote source, e.g. via the internet, are provided. An example ofthis embodiment is a kit that includes a web address where theinstructions can be viewed and/or from which the instructions can bedownloaded. As with the instructions, this means for obtaining theinstructions is recorded on a suitable substrate.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); nt,nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c.,subcutaneous(ly); and the like.

Materials and Methods

The following methods and materials were used in the Examples below.

Animals. C57BL/6 mice were purchased from the Charles River Laboratory(Wilmington, Mass.), Mice were kept in accordance with welfareguidelines and project license restrictions under controlled light (12hr light and 12 hr dark cycle, dark 6:30 pm-6:30 am), temperature (22±4°C.) and humidity (50%±20%) conditions. They had free access to water(autoclaved distilled water) and were fed ad libitum on a commercialdiet (Harlan laboratories, Irradiated 2018 Teklad Global 18% ProteinRodent Diet) containing 17 kcal % fat, 23 kcal % protein and 60 kcal %carbohydrate. Alternatively, mice were maintained on a high-fat diet(D12492, Research Diets, New Brunswick, N.J. USA) containing 60 kcal %fat, 20 kcal % protein and 20 kcal % carbohydrate. All animal studieswere approved by the NGM Institutional Animal Care and Use Committee forNGM-5-2008 entitled “Characterization Of Biologics, Compounds And ViralVectors For Treatment Of Diabetes Using Rodent Model”.

DNA and amino acid sequences.

cDNA of ORF encoding murine FAM3C (GenBank Accession No. NM_138587.4)(SEQ ID NO: 9) ATGAGGGTAGCAGGAGCTGCAAAGTTGGTAGTGGCCGTGGCAGTATTCTTACTGACCTTCTATGTTATTTCTCAAGTATTTGAAATTAAAATGGATGCAAGTTTAGGAAATCTATTTGCTCGATCCGCGCTGGACTCAGCCATTCGTTCTACGAAACCTCCGAGGTACAAGTGTGGGATCTCAAAGGCGTGCCCAGAGAAGCATTTTGCTTTTAAGATGGCTAGTGGAGCAGCCAATGTCGTGGGACCCAAGATCTGCCTGGAGGACAATGTTTTGATGAGTGGTGTGAAGAATAATGTCGGAAGAGGAATCAATATTGCCTTGGTAAATGGGAAAACAGGGGAAGTAATAGACACCAAATTTTTTGACATGTGGGGAGGAGATGTGGCACCATTCATTGAGTTTTTGAAGACCATACAAGACGGAACAGTAGTGCTAATGGCTACATACGATGATGGAGCAACCAAACTCACGGATGAGGCACGGCGGCTCATTGCTGAACTGGGCAGCACTTCGATCACCAGTCTTGGTTTCCGAGATAACTGGGTCTTCTGTGGTGGGAAGGGCATTAAGACAAAGAGTCCCTTTGAACAGCACATAAAGAACAATAAGGAAACGAACAAGTACGAGGGATGGCCTGAGGTGGTGGAGATGGAAGGATGTATCCCCCAG AAGCAAGACTGA.Protein sequence encoded by the cDNA (GenBank Accession No. NP_613053.3)(SEQ ID NO: 10) MRVAGAAKLVVAVAVFLLTFYVISQVFEIKMDASLGNLFARSALDSAIRSTKPPRYKCGISKACPEKHFAFKMASGAANVVGPKICLEDNVLMSGVKNNVGRGINIALVNGKTGEVIDTKFFDMWGGDVAPFIEFLKTIQDGTVVLMATYDDGATKLTDEARRLIAELGSTSITSLGFRDNWVFCGGKGIKTKSPFEQHIKNNKETNKYEGWPEVVEMEGCIPQKQD.

FAM3C ORF was amplified with polymerase chain reaction (PCR) usingrecombinant DNA (cDNA) prepared from mouse small intestinal tissue. PCRreagents kits with Phusion high-fidelity DNA polymerase were purchasedfrom New England BioLabs (F-530L, Ipswich, Mass.). The following primerswere used: forward PCR primer: 5′ ATGAGGGTAGCAGGAGCTGCA (SEQ ID NO: 11)and reverse PCR primer: 5′ TCAGTCTTGCTTCTGGGGGAT (SEQ ID NO: 12).

PCR.

The PCR reaction were set up according to manufacturer's instruction,amplified DNA fragment was digested with restriction enzymes Spe I andNot I (the restriction sites were included in the 5′ or 3′ PCR primers,respectively) and was then ligated with AAV transgene vectors that hadbeen digested with the same restriction enzymes. The vector used forexpression contained a selectable marker and an expression cassettecomposed of a strong eukaryotic promoter 5′ of a site for insertion ofthe cloned coding sequence, followed by a 3′ untranslated region andbovine growth hormone polyadenylation tail. The expression construct isalso flanked by internal terminal repeats at the 5′ and 3′ ends.

Production and purification of AAV. AAV 293 cells (obtained from AgilentTechnologies, Santa Clara, Calif.) were cultured in Dulbeco'sModification of Eagle's Medium (DMEM, Mediatech, Inc. Manassas, Va.)supplemented with 10% fetal bovine serum and 1× antibiotic-antimycoticsolution (Mediatech, Inc. Manassas, Va.). The cells were plated at 50%density on day 1 in 150 mm cell culture plates and transfected on day 2,using calcium phosphate precipitation method, the following 3 plasmids(20 μg/plate of each): AAV transgene plasmid, pHelper plasmids (AgilentTechnologies) and AAV2/9 plasmid (Gao et al (2004) J. Virol. 78:6381).48 hours after transfection, the cells were scraped off the plates,pelleted by centrifugation at 3000×g and resuspended in buffercontaining 20 mM Tris pH 8.5, 100 mM NaCl and 1 mM MgCl₂. The suspensionwas frozen in an alcohol dry ice bath and was then thawed in 37° C.water bath. The freeze and thaw cycles were repeated for a total ofthree times; benzonase (Sigma-Aldrich, St. Louis, Mo.) was added to 50units/ml; deoxycholate was added to a final concentration of 0.25%.After an incubation at 37° C. for 30 min, cell debris was pelleted bycentrifugation at 5000×g for 20 min. Viral particles in the supernatantwere purified using a discontinued iodixanal (Sigma-Aldrich, St. Louis,Mo.) gradient as previously described (Zolotukhin S. et al (1999) GeneTher 6:973). The viral stock was concentrated using Vivaspin 20 (MWcutoff 100,000 Dalton, Sartorius Stedim Biotech, Aubagne, France) andre-suspended in phosphate-buffered saline (PBS) with 10% glycerol andstored at −80° C. To determine the viral genome copy number, 2 μl ofviral stock was incubated in 6 μl of solution containing 50 units/mlbenzonase, 50 mM Tris-HCl pH 7.5, 10 mM MgCl₂ and 10 mM CaCl₂ for at 37°C. for 30 minutes.

Afterwards, 15 μl of the solution containing 2 mg/ml of Proteinase K,0.5% SDS and 25 mM EDTA were added and the mixture was incubated foradditional 20 min at 55° C. to release viral DNA. Viral DNA was cleanedwith mini DNeasy Kit (Qiagen, Valencia, Calif.) and eluted with 40 μl ofwater. Viral genome copy (GC) was determined by using quantitative PCR.

Viral stock was diluted with PBS to desirable GC/ml. 200 μl of viralworking solution was delivered into mice via tail vein injection.

Blood Glucose Assay.

Blood glucose in mouse tail snip was measured using ACCU-CHEK Activetest strips read by an ACCU-CHEK Active meter (Roche Diagnostics,Indianapolis, Ind.) following manufacturer's instruction.

Serum Insulin Assay.

Whole blood (about 50 μl/mouse) from mouse tail snips was collected intoplain capillary tubes (BD Clay Adams SurePrep, Becton Dickinson and Co.Sparks, Md.). Serum and blood cells were separated by spinning the tubesin an Autocrit Ultra 3 (Becton Dickinson and Co. Sparks, Md.). Insulinlevels in serum were determined using insulin EIA kits (80-Insums-E01,Alpco Diagnostics, Salem, N.H.) by following manufacturer's instruction.

Glucose Tolerance Test (GTT).

Mice fasted for 16 hours received glucose (1 g/kg) in PBS viaintra-peritoneal injection. Blood glucose levels were determined asdescribed above at the time points indicated.

Insulin Tolerance Test (ITT).

Mice fasted for 4 hours received 0.75 units/kg of insulin (Humulin R EliLilly and Co. Indianapolis, Ind.) via intra-peritoneal injection. Bloodglucose was determined as described above.

Statistics.

Statistical analysis was performed with Student's t-Test with 2-taileddistribution.

Example 1 Effect of in Vivo FAM3C Expression on Blood Glucose Levels inMice with Diet-Induced Obesity

To identify secreted proteins that have an effect on glucose metabolism,selected genes were overexpressed in mice using adeno-associated virus(AAV) as the gene delivery vehicle. The anti-diabetic effects of thegene products were evaluated in diet-induced obesity (DIO) model. Eightweek old male C57BL/6 mice were subjected to 60% kcal fat diet for eightweeks before they received a one-time tail vein injection of recombinantAAV (rAAV). Mice body weight, blood glucose and serum insulin weredetermined. Glucose tolerance and insulin tolerance tests were alsoperformed to help assess the effect of rAAV on glucose clearance andinsulin sensitivity. rAAV-mediated Fam3C expression significantlyreduced blood glucose levels in DIO mice without significantly changingthe body weight (FIG. 1). Results of the glucose tolerance testindicated improvement of glucose disposal in these animals.

The ability of murine Fam3C to regulate the level of plasma glucose wastested as follows. rAAV expressing Fam3C was injected through tail veininto mice that had been on high fat diet for eight weeks. Prior to, andat two and four weeks after the injection, 4-hour fasting blood glucoselevels were determined in tail blood. In FIG. 2, “Chow” refers to miceon chow diet, “GFP” to DIO mice that were injected with 1×10¹² genomecopies (“1E+12” “GC”) of rAAV expressing green fluorescent protein, and“Fam3C” to mice injected with 1E+12GC of rAAV expressing Fam3C (n=5 miceper group). As seen in FIG. 2, recombinant AAV expressing murine Fam3Creduced blood glucose in DIO mice to levels comparable to mice on chowdiet.

Example 2 Effect of Murine FAM3C Expression on Serum Insulin Levels inMice with Diet-Induced Obesity

The ability of murine Fam3C to relieve hyperinsulinemia in mice withdiet-induced obesity was tested. rAAV was injected through tail veininto mice that had been on high fat diet for eight weeks. At the twoweek and four week time points after the AAV injection, tail blood wascollected from mice that had been fasting for four hours, and seruminsulin were determined by enzyme-linked immunosorbent assay (ELISA). InFIG. 3, “Chow” refers to mice on chow diet; “GFP” to DIO mice that wereinjected with 1E+12 GC of rAAV expressing green fluorescent protein, and“Fam3C” to mice injected with 1E+12 GC of rAAV expressing Fam3C (n=5mice per group). As seen in FIG. 3, recombinant AVV expressing murineFam3C reduced hyperinsulinemia in DIO mice at the four week time point.

Example 3 Effect of Murine FAM3C Expression on Glucose Tolerance in Micewith Diet-Induced Obesity

The ability of murine Fam3C to improve glucose tolerance of mice withdiet-induced obesity was evaluated as follows. rAAV expressing Fam3C wasinjected through tail vein into mice that had been on high fat diet foreight weeks. Glucose tolerance test was performed three weeks after theAAV injection. Mice fasted overnight received 1 g/kg of glucose in PBSvia intraperitoneal injection (i.p.). Blood glucose levels weredetermined at times indicated. In FIG. 4, “Chow” refers to mice on chowdiet, “GFP” to DIO mice that were injected with 1E+12 GC of rAAVexpressing green fluorescent protein, and “Fam3C” to mice injected with1E+12 GC of rAAV expressing Fam3C (n=5 mice per group). As seen in FIG.4, recombinant AAV expressing murine Fam3C was able to improve glucosetolerance in DIO mice.

Example 4 Effect of Murine FAM3C Expression on Insulin Tolerance in Micewith Diet-Induced Obesity

The ability of murine Fam3C to improve insulin sensitivity of mice withdiet-induced obesity was evaluated as follows. rAAV expressing Fam3C wasinjected through tail vein into mice that had been on high fat diet foreight weeks. An insulin tolerance test was performed five weeks afterthe AAV injection. Glucose levels were monitored after anintraperitoneal injection of insulin (0.75 units/kg). Response toinsulin was compared among DIO mice injected with AAV expressing Fam3C,GFP and lean mice by measuring blood glucose levels at times indicated.In FIG. 5, “Chow” refers to mice on chow diet, “GFP” to DIO mice thatwere injected with 1E+12 GC of rAAV expressing green fluorescentprotein, and “Fam3C” to mice injected with 1E+12 GC of rAAV expressingFam3C (n=5 mice per group). As seen in FIG. 5, recombinant AAVexpressing murine Fam3C was able to improve insulin sensitivity in DIOmice.

Example 5 Cloning of the Human Gene

The cloning of the human gene encoding FAM3C is carried out by using thePCR method as previously set forth for the cloning of the mouse gene.Briefly, the human FAM3C gene can be cloned out by PCR from cDNA libraryusing the following pair of primers, and then cloned into AAV transgenevector as described above for efficacy evaluation. Forward PCR primer:5′ ATGAGGGTAGCAGGTGCTGCA (SEQ ID NO: 13). Reverse PCR primer: 5′TTAGTCTTGCTTCTGGGGGAT (SEQ ID NO: 14).

The nucleic acid sequences, and the encoded amino acid sequence, forhuman FAM3C are provided below:

Human FAM3C ORF (GenBank Accession No. NM_014888.2) (SEQ ID NO: 15)ATGAGGGTAGCAGGTGCTGCAAAGTTGGTGGTAGCTGTGGCAGTGTTTTTACTGACATTTTATGTTATTTCTCAAGTATTTGAAATAAAAATGGATGCAAGTTTAGGAAATCTATTTGCAAGATCAGCATTGGACACAGCTGCACGTTCTACAAAGCCTCCCAGATATAAGTGTGGGATCTCAAAAGCTTGCCCTGAGAAGCATTTTGCTTTTAAAATGGCAAGTGGAGCAGCCAACGTGGTGGGACCCAAAATCTGCCTGGAAGATAATGTTTTAATGAGTGGTGTTAAGAATAATGTTGGAAGAGGGATCAATGTTGCCTTGGCAAATGGAAAAACAGGAGAAGTATTAGACACTAAATATTTTGACATGTGGGGAGGAGATGTGGCACCATTTATTGAGTTTCTGAAGGCCATACAAGATGGAACAATAGTTTTAATGGGAACATACGATGATGGAGCAACCAAACTCAATGATGAGGCACGGCGGCTCATTGCTGATTTGGGGAGCACATCTATTACTAATCTTGGTTTTAGAGACAACTGGGTCTTCTGTGGTGGGAAGGGCATTAAGACAAAAAGCCCTTTTGAACAGCACATAAAGAACAATAAGGATACAAACAAATATGAAGGATGGCCTGAAGTTGTAGAAATGGAAGGATGCATCCCCCAG AAGCAAGACTAA.Human FAM3C 227 amino acid residues (GenBank Accession No. NP_055703.1)(SEQ ID NO: 16) MRVAGAAKLVVAVAVFLLTFYVISQVFEIKMDASLGNLFARSALDTAARSTKPPRYKCGISKACPEKHFAFKMASGAANVVGPKICLEDNVLMSGVKNNVGRGINVALANGKTGEVLDTKYFDMWGGDVAPFIEFLKAIQDGTIVLMGTYDDGATKLNDEARRLIADLGSTSITNLGFRDNWVFCGGKGIKTKSPFEQHIKNNKDTNKYEGWPEVVEMEGCIPQKQD.

Example 6 Treatment of Mice with Diet-Induced Obesity with rAAVExpressing Human FAM3C

The human gene will be cloned into AAV transgene vector. Recombinant AAVexpressing the corresponding proteins will be generated as describedabove in Materials and Methods.

The ability of human FAM3C (hFAM3C) to regulate the level of plasmaglucose can be tested as follows. rAAV is injected through tail veininto mice that have been on high fat diet for eight weeks. Two weeksafter the injection, 4-hour fasting blood glucose levels are determinedin tail bleed using a glucometer. Mice tested include a lean group ofmice on chow diet (“Chow”), a “GFP” group of DIO mice that are injectedwith 1E+12 GC of rAAV expressing green fluorescent protein, and a“hFAM3C” group of DIO mice injected with 1E+12GC of rAAV expressinghFAM3C (n=5 mice per group).

The ability of hFAM3C to relieve hyperinsulinemia in mice withdiet-induced obesity can also be tested. rAAV is injected through tailvein into mice that have been on high fat diet for eight weeks. Before,and at two and four weeks after the AAV injection, tail blood iscollected from mice that have been fasting for four hours, and seruminsulin is determined by ELISA. Groups of mice tested can include a leangroup of mice on chow diet (“Chow”), a “GFP” group of DIO mice that areinjected with 1E+12 GC of rAAV expressing green fluorescent protein, anda “hFAM3C” group of DIO mice injected with 1E+12 GC of rAAV expressinghFAM3C (n=5 mice per group).

The ability of hFAM3C to improve glucose tolerance of mice withdiet-induced obesity can be evaluated as follows. rAAV is injectedthrough tail vein into mice that have been on high fat diet for eightweeks. Glucose tolerance test is performed three weeks after the AAVinjection. Mice fasted overnight are injected with 1 g/kg of glucose inPBS via intraperitoneal injection (i.p.). Blood glucose levels aredetermined at various timed intervals. Groups of mice under evaluationinclude a group of lean mice on chow diet (“Chow”), a “GFP” group of DIOmice that are injected with 1E+12 GC of rAAV expressing greenfluorescent protein, and a“hFAM3C” group of DIO mice injected with1E+12GC of rAAV expressing hFAM3C (n=5 mice per group).

Example 7 Expression of Recombinant Murine and Human FAM3C

For recombinant protein expression in the mammalian expression systems,the cDNA sequence encoding the murine or human FAM3C is cloned intoNheI/MluI or NheI/XbaI sites of a modified pCDNA3.1 vector, so that theexpressed protein is tagged with either 6× His or human Fc. Aftersequence confirmation, the plasmid is tested for expression andsecretion by transient transfection of the plasmids into suspension-,serum-free adapted 293T, 293-F, and CHO-S cells using FreeStyle MAXtransfection reagent (Invitrogen). The identity of the secreted proteinis confirmed by anti-His, Anti-hFc, and/or available gene-specificantibodies. The cell line revealing the highest level of the proteinsecretion is then selected for large-scale transient production of theprotein in spinners and/or Wave Bioreactor® System for 5-7 days. Therecombinant protein in the supernatant from the transient production ispurified by Ni-NTA beads or Protein A-Sepharose affinity chromatographyusing ÄKTAexplorer™ (GE Healthcare), and followed by other purificationmethods, if needed. The purified protein is then dialyzed against PBS,concentrated to ˜1 mg/ml or higher concentrations, and stored at −80° C.until use.

For recombinant protein expression in the bacterial expression system,the cDNA sequence encoding the FAM3C protein is cloned into NdeI/HindIII or KpnI/Hind III sites of pET30(+) vector, so that the expressedprotein is tagged with 6× His. The sequencing confirmed plasmid istransformed into BL21(DE3) cells. The protein expression is induced byadding IPTG in the culture and confirmed with anti-His or gene-specificantibodies. If the expressed protein is in the soluble fraction, it willbe purified by Ni-NTA affinity chromatography followed by otherpurification methods if needed. If the expressed protein is in inclusionbodies, the inclusion bodies will be isolated first. The protein in theinclusion bodies is denatured using urea or other denaturing reagents,purified by Ni-NTA beads, refolded, and further purified using othermethods if needed. Endotoxin level in the purified protein is thenexamined, and removed by different methods until the endotoxin level iswithin the acceptable range. The protein is then dialyzed, concentratedand stored as described above.

Example 8 Treatment of Mice with Diet-Induced Obesity with Human FAM3CRecombinant Protein

The ability of murine and human FAM3C to regulate the level of plasmaglucose can be tested as follows. Recombinant murine or human FAM3Cprotein and control protein dissolved in PBS is injected into mice onhigh-fat diet at 30, 10, and 3 mg/kg via IP, SC or IV once a day for twoweeks. Body weight, 4-hour fasting blood glucose levels are determinedone and two weeks after the initiation of injections. Glucose tolerancetest is carried out performed in week 2 and serum insulin is alsodetermined in week 2. Assays are performed as described above inExamples 1-4.

Example 9 Effect of in Vivo Human FAM3C Expression on Blood GlucoseLevels in Mice with Diet-Induced Obesity

The anti-diabetic effect of human FAM3C was evaluated in the DIO mousemodel described above. As described in Example 1, eight-week-old maleC57BL/6 mice were subjected to 60% kcal fat diet for eight weeks beforethey received a one-time tail vein injection of rAAV comprising anucleotide sequence encoding human FAM3C. Mice body weight, bloodglucose, and serum insulin were determined. Glucose tolerance andinsulin tolerance tests were also performed to help assess the effect ofrAAV on glucose clearance and insulin sensitivity. rAAV-mediated humanFAM3C expression significantly reduced body weight, blood glucose, andserum insulin levels in DIO mice (FIGS. 6-8). Results of the glucose andinsulin tolerance tests indicated improvement of glucose disposal andinsulin sensitivity in these animals (FIGS. 9-10).

The ability of human FAM3C to regulate the level of plasma glucose wastested as follows. rAAV expressing human FAM3C was injected through thetail vein into mice that had been on high fat diet for eight weeks. Twoweeks after the injection, 4-hour fasting blood glucose levels weredetermined in tail blood. In FIG. 7, “Chow” refers to lean mice on chowdiet, “GFP” to DIO mice that were injected with 1×10¹² genome copies(“1E+12” “GC”) of rAAV expressing green fluorescent protein (GFP),“hFAM3C” to mice injected with 1E+12GC of rAAV expressing human FAM3C(n=5 mice per group). As seen in FIG. 7, recombinant AAV expressinghuman FAM3C reduced blood glucose in DIO mice.

Example 10 Effect of Human FAM3C Expression on Serum Insulin Levels inMice with Diet-Induced Obesity

The ability of human FAM3C to relieve hyperinsulinemia in mice withdiet-induced obesity was tested. rAAV expressing human FAM3C wasinjected through the tail vein into mice that had been on high fat dietfor eight weeks. At the two and four week time points after the AAVinjection, tail blood was collected from mice that had been fasting forfour hours, and serum insulin was determined by ELISA. In FIG. 8, “Chow”refers to lean mice on chow diet; “GFP” to DIO mice that were injectedwith 1E+12 GC of rAAV expressing green fluorescent protein, and “hFAM3C”to mice injected with 1E+12 GC of rAAV expressing human FAM3C (n=5 miceper group). As seen in FIG. 8, recombinant AAV expressing human FAM3Crelieved hyperinsulinemia in DIO mice.

Example 11 Effect of Human FAM3C Expression on Glucose Tolerance in Micewith Diet-Induced Obesity

The ability of human FAM3C to improve glucose tolerance of mice withdiet-induced obesity was evaluated as follows. rAAV expressing humanFAM3C was injected through the tail vein into mice that had been on highfat diet for eight weeks. A glucose tolerance test was performed threeweeks after the AAV injection. Mice fasted overnight received 1 g/kg ofglucose in phosphate buffered saline (PBS) via intraperitoneal (i.p.)injection. Blood glucose levels were determined at times indicated. InFIG. 9, “Chow” refers to lean mice on chow diet; “GFP” refers to DIOmice that were injected with 1E+12 GC of rAAV expressing greenfluorescent protein, and “hFAM3C” to mice injected with 1E+12 GC of rAAVexpressing human FAM3C (n=5 mice per group). As seen in FIG. 9,recombinant AAV expressing human FAM3C was able to improve glucosetolerance in DIO mice.

Example 12 Effect of Human FAM3C Expression on Insulin Sensitivity inMice with Diet-Induced Obesity

The ability of human FAM3C to improve insulin sensitivity of mice withdiet-induced obesity was evaluated as follows. rAAV expressing humanFAM3C was injected through the tail vein into mice that had been on highfat diet for eight weeks. An insulin tolerance test was performed fiveweeks after the AAV injection. Glucose levels were monitored after anintraperitoneal injection of insulin (0.75 units/kg). Response toinsulin was compared among DIO mice injected with AAV expressing humanFAM3C and GFP by measuring blood glucose levels at times indicated. InFIG. 10 “GFP” refers to DIO mice that were injected with 1E+12 GC ofrAAV expressing green fluorescent protein, and “hFAM3C” to mice injectedwith 1E+12 GC of rAAV expressing human FAM3C (n=5 mice per group). Asseen in FIG. 10, recombinant AAV expressing human FAM3C was able toimprove insulin sensitivity in DIO mice.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. A method of treating a subject comprising:administering to said subject having a glucose metabolism disorder atherapeutically effective amount of a protein comprising at least 90%amino acid sequence identity to an amino acid sequence of human FAM3C,wherein said administering is effective to treat a symptom of theglucose metabolism disorder.
 2. The method of claim 1, wherein saidglucose metabolism disorder comprises hyperglycemia and wherein saidadministering reduces plasma glucose in said subject.
 3. The method ofclaim 1, wherein said glucose metabolism disorder compriseshyperinsulinemia and wherein said administering reduces plasma insulinin said subject.
 4. The method of claim 1, wherein said glucosemetabolism disorder comprises glucose intolerance and wherein saidadministering increases glucose tolerance in said subject.
 5. The methodof claim 1, wherein said glucose metabolism disorder comprises diabetesmellitus.
 6. The method of claim 1, wherein said subject is obese. 7.The method of claim 1, wherein said glucose metabolism disorder isdiet-induced.
 8. The method of claim 1, wherein said subject is human.9. The method of claim 1, wherein said administering is by parenteralinjection.
 10. The method of claim 9, wherein said parenteral injectionis subcutaneous.
 11. The method of claim 1, wherein said protein isadministered in an amount of from about 0.1 μg/kg to about 1 mg/kg. 12.The method of claim 1, wherein said protein is administered in an amountof from about 0.1 μg/kg to about 1 μg/kg.